Golden rice is a variety of Oryza sativa (rice) produced through genetic engineering of biosynthesized beta-carotene, a precursor of Vitamin-A in the edible parts of rice developed by Ingo Potrykus and his group. The aim is to produce a fortified food to be grown and consumed in areas with a shortage of dietary Vitamin-A, which kills so many children under five year age. Golden rice differs from its parental strain by the addition of three beta-carotene biosynthesis genes namely 'psy' (phytoene synthase) from daffodil plant Narcissus pseudonarcissus and 'crt-I' gene from the soil bacterium Erwinia uredovora and 'lyc' (lycopene cyclase) gene from wild-type rice endosperm. The endosperm of normal rice, does not contain beta-carotene. Golden-rice has been genetically altered so that the endosperm now accumulates Beta-carotene. This has been done using Recombinant DNA technology. Golden rice can control childhood blindness - Xerophthalmia.

Bt cotton is a genetically modified organism (GMO) or genetically modified pest resistant plant cotton variety, which produces an insecticide activity to bollworm. Strains of the bacterium Bacillus thuringiensis produce over 200 different Bt toxins, each harmful to different insects. Most Bt toxins are insecticidal to the larvae of moths and butterflies, beetles, cotton bollworms and gat flies but are harmless to other forms of life. The genes are encoded for toxic crystals in the Cry group of endotoxin. When insects attack and eat the cotton plant the Cry toxins are dissolved in the insect's stomach. The epithelial membranes of the gut block certain vital nutrients thereby sufficient regulation of potassium ions are lost in the insects and results in the death of epithelial cells in the intestine membrane which leads to the death of the larvae.

Introduction of 'bar' gene through vector
                      \(\downarrow \)
Cell culture of potato with 'bar' gene
                     \(\downarrow \)
Herbicide tolerant potato cells
                      \(\downarrow \)
In vitro culture
                       \(\downarrow \)
Callus ~ Organogenesis
                       \(\downarrow \)
Development of Herbicide tolerant transgenic plants

The shuttle vectors are plasmids designed to replicate in cells of two different species. These vectors are created by recombinant techniques. The shuttle vectors can propagate in one host and then move into another host without any extra manipulation. Most of the Eukaryotic vectors are Shuttle Vectors

(i) Transposons (Transposable elements or mobile elements) are DNA sequence able to insert itself at a new location in the genome without having any sequence relationship with the target locus and hence transposons are called walking genes or jumping genes.
(ii) They are used as genetic tools for analysis of gene and protein functions, that produce new phenotype on host cell.
(iii) The use of transposons is well studied in Arabidopsis thaliana and bacteria such as Escherichia coli.

Properties of Vectors:
1. Vectors are able to replicate autonomously to produce multiple copies of them along with their DNA insert in the host cell.
2. It should be small in size and of low molecular weight, less than 10 Kb (kilo base pair) in size so that entry/transfer into host cell is easy.
3. Vector must contain an origin of replication so that it can independently replicate within the host.
4. It should contain a suitable marker such as antibiotic resistance, to permit its detection in transformed host cell.
5. Vector should have unique target sites for integration with DNA insert and should have the ability to integrate with DNA insert it carries into the genome of the host cell. Most of the commonly used cloning vectors have more than one restriction site. These are Multiple Cloning Site (MCS) or polylinker. Presence of MCS facilitates the use of restriction enzyme of choice.

A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites.
Types
(i) Exonucleases - which remove nucleotides one at a time from the end of a DNA molecule. e.g. Bal 31, Exonuclease III.
(ii) Endonucleases - which break the internal phosphodiester bonds within a DNA molecule. e.g. Hind II, EcoRI, PvulI, BamHI, TaqI.
(iii) The restriction enzymes are called as molecular scissors
(iv) They are used in recombinant DNA technology as they recognize & cut DNA within a specific sequence.

(i) pBR 322 plasmid is a reconstructed plasmid and most widely used as cloning vector;
(ii) It contains 4361 base pairs. In pBR, p denotes plasmid, Band R respectively the names of scientist Boliver and Rodriguez who developed this plasmid.
(iii) The number 322 is the number of plasmid developed from their laboratory.
(iv) It contains amp^R and tet^R two different antibiotic resistance genes and recognition sites for several restriction enzymes.
(v) ori and antibiotic resistance genes. Rop codes for the proteins involved in the replication of the plasmid.

Direct or Vector less Gene Transfer
In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant without the help of a vector, The following are some of the common methods of direct gene transfer in plants.
(i) Chemical mediated gene transfer: Certain chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.
(ii) Microinjection: The DNA is directly injected into the nucleus using fine tipped glass needle or micro pipette to transform plant cells. The protoplasts are immobilized on a solid support (agarose on a microscopic slide) or held with a holding pipette under section.
(iii) Electroporation Methods of Gene Transfer: A pulse of high voltage is applied to protoplasts, cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs.

(iv) Liposome mediated method of Gene transfer: Liposomes the artificial phospholipid vesicles are useful in gene transfer. The gene or DNA is transferred from liposome into vacuole of plant cells. It is carried out by encapsulating DNA into the vacuole. This technique is advantageous because the liposome protects the introduced DNA from being damaged by the acidic pH and protease enzymes present in the vacuole. Liposome and tonoplast of  vacuole fusion resulted in gene transfer. This process is called lipofection.

(v) Biolistics: The foreign DNA is coated onto the surface of minute gold or tungsten particles (1-3 \(\mu\)m) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/ micro projectile gun/shotgun) Then the bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells.

Applications of Biotechnology
1. Biotechnology is one of the most important applied interdisciplinary sciences of the 21st century. It is the trusted area that enables us to find the beneficial way of life.
2. Biotechnology has wide applications in various sectors like agriculture, medicine, environment and commercial industries.
3. This science has an invaluable outcome like transgenic varieties of plants e.g. transgenic cotton (Bt-cotton), rice, tomato, tobacco, cauliflower, potato and banana.
4. The development of transgenics as pesticide resistant, stress resistant and disease resistant varieties of agricultural crops is the immense outcome of biotechnology.
5. The synthesis of human insulin and blood protein in E.coli and utilized for insulin deficiency disorder in human is a breakthrough in biotech industries in medicine.
6. The synthesis of vaccines, enzymes, antibiotics, dairy products and beverages are the products of biotech industries.
7. Biochip based biological computer is one of the successes of biotechnology.
8. Genetic engineering involves genetic manipulation, tissue culture involves aseptic cultivation of totipotent plant cell into plant clones under controlled atmospheric conditions.
9. Single cell protein from Spirulina is utilized in food industries.
10. Production of secondary metabolites, biofertilizers, biopesticides and enzymes.
11. Biomass energy, biofuel, bioremediation and phytoremediation for environmental biotechnology.